Base and nucleotide excision repair facilitate resolution of platinum drugs-induced transcription blockage

Sensitivity and resistance of cells to platinum drug chemotherapy are to a large extent determined by activity of the DNA damage response (DDR). Combining chemotherapy with inhibition of specific DDR pathways could therefore improve treatment efficacy. Multiple DDR pathways have been implicated in removal of platinum-DNA lesions, but it is unclear which exact pathways are most important to cellular platinum drug resistance. Here, we used CRISPR/Cas9 screening to identify DDR proteins that protect colorectal cancer cells against the clinically applied platinum drug oxaliplatin. We find that besides the expected homologous recombination, Fanconi anemia and translesion synthesis pathways, in particular also transcription-coupled nucleotide excision repair (TC-NER) and base excision repair (BER) protect against platinum-induced cytotoxicity. Both repair pathways are required to overcome oxaliplatin- and cisplatin-induced transcription arrest. In addition to the generation of DNA crosslinks, exposure to platinum drugs leads to reactive oxygen species production that induces oxidative DNA lesions, explaining the requirement for BER. Our findings highlight the importance of transcriptional integrity in cells exposed to platinum drugs and suggest that both TC-NER and BER should be considered as targets for novel combinatorial treatment strategies

The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development

FIRRM/C1orf112 is synthetic lethal with PICH and mediates RAD51 dynamics

Joint DNA molecules are natural byproducts of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, compromising sister chromatid separation. The DNA translocase PICH (ERCC6L) has a central role in UFB resolution. A genome-wide loss-of-function screen is performed to identify the genetic context of PICH dependency. In addition to genes involved in DNA condensation, centromere stability, and DNA-damage repair, we identify FIGNL1-interacting regulator of recombination and mitosis (FIRRM), formerly known as C1orf112. We find that FIRRM interacts with and stabilizes the AAA + ATPase FIGNL1. Inactivation of either FIRRM or FIGNL1 results in UFB formation, prolonged accumulation of RAD51 at nuclear foci, and impaired replication fork dynamics and consequently impairs genome maintenance. Combined, our data suggest that inactivation of FIRRM and FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in persistent DNA lesions and a dependency on PICH to preserve cell viability. </p

The learning curve of transanal total mesorectal excision for rectal cancer is associated with local recurrence: results from a multicentre external audit: results from a multicentre external audit

Aim: Transanal total mesorectal excision (TaTME) has been suggested as a potential solution for the resection of challenging mid and low rectal cancer. This relatively complex procedure has been implemented in many centres over the last years, despite the absence of long-term safety data. Recently, concern has arisen because of an increase in local recurrence in the implementation phase. The aim of this study was to assess the correlation between accumulated experience and local recurrences. Method: An independent clinical researcher performed an external audit of consecutive series of all TaTME procedures in six centres in the Netherlands. Kaplan–Meier estimated local recurrence rates were calculated and multivariate Cox proportional hazards regression analysis performed to assess risk factors for local recurrence. Primary outcome was the local recurrence rate in the initial implementation (cases 1–10), continued adoption (cases 11-40) and prolonged experience (case 41 onward). Results: Six hundred and twenty-four consecutive patients underwent TaTME for rectal cancer with a median follow-up of 27 months (range 1–82 months). The estimated 2- and 3-year local recurrence rates were 4.6% and 6.6%, respectively. Cox proportional hazards regression revealed procedural experience to be an independent factor in multivariate analysis next to advanced stage (ycMRF+, pT3-4, pN+) and pelvic sepsis. Corrected analysis projected the 3-year local recurrence rates to be 9.7%, 3.3% and 3.5% for the implementation, continued adoption and prolonged experience cohorts, respectively. Conclusion: This multicentre study shows a high local recurrence rate (12.5%) after implementation of TaTME which lowers to an acceptable rate (3.4%) when experience increases. Therefore, intensified proctoring and further precautions must be implemented to reduce the unacceptably high risk of local recurrence at units starting this technique

Sister chromatid exchanges induced by perturbed replication can form independently of BRCA1, BRCA2 and RAD51

Sister chromatid exchanges (SCEs) are products of joint DNA molecule resolution, and are considered to form through homologous recombination (HR). Indeed, SCE induction upon irradiation requires the canonical HR factors BRCA1, BRCA2 and RAD51. In contrast, replication-blocking agents, including PARP inhibitors, induce SCEs independently of BRCA1, BRCA2 and RAD51. PARP inhibitor-induced SCEs are enriched at difficult-to-replicate genomic regions, including common fragile sites (CFSs). PARP inhibitor-induced replication lesions are transmitted into mitosis, suggesting that SCEs can originate from mitotic processing of under-replicated DNA. Proteomics analysis reveals mitotic recruitment of DNA polymerase theta (POLQ) to synthetic DNA ends. POLQ inactivation results in reduced SCE numbers and severe chromosome fragmentation upon PARP inhibition in HR-deficient cells. Accordingly, analysis of CFSs in cancer genomes reveals frequent allelic deletions, flanked by signatures of POLQ-mediated repair. Combined, we show PARP inhibition generates under-replicated DNA, which is processed into SCEs during mitosis, independently of canonical HR factors